Assembly of the herpes simplex virus capsid: preformed triplexes bind to the nascent capsid

Abstract

The herpes simplex virus type 1 (HSV-1) capsid is a T=16 icosahedral shell that forms in the nuclei of infected cells. Capsid assembly also occurs in vitro in reaction mixtures created from insect cell extracts containing recombinant baculovirus-expressed HSV-1 capsid proteins. During capsid formation, the major capsid protein, VP5, and the scaffolding protein, pre-VP22a, condense to form structures that are extended into procapsids by addition of the triplex proteins, VP19C and VP23. We investigated whether triplex proteins bind to the major capsid-scaffold protein complexes as separate polypeptides or as preformed triplexes. Assembly products from reactions lacking one triplex protein were immunoprecipitated and examined for the presence of the other. The results showed that neither triplex protein bound unless both were present, suggesting that interaction between VP19C and VP23 is required before either protein can participate in the assembly process. Sucrose density gradient analysis was employed to determine the sedimentation coefficients of VP19C, VP23, and VP19C-VP23 complexes. The results showed that the two proteins formed a complex with a sedimentation coefficient of 7.2S, a value that is consistent with formation of a VP19C-VP23(2) heterotrimer. Furthermore, VP23 was observed to have a sedimentation coefficient of 4.9S, suggesting that this protein exists as a dimer in solution. Deletion analysis of VP19C revealed two domains that may be required for attachment of the triplex to major capsid-scaffold protein complexes; none of the deletions disrupted interaction of VP19C with VP23. We propose that preformed triplexes (VP19C-VP23(2) heterotrimers) interact with major capsid-scaffold protein complexes during assembly of the HSV-1 capsid.

FIG. 1

Protein compositions of products formed in the in vitro assembly system. Cell extracts containing recombinant VP5, pre-VP22a, VP23, and VP19C (ALL) were combined and assembly products were precipitated with the VP5-specific monoclonal antibody 6F10. The precipitate was analyzed by SDS-PAGE, and the gel was stained with Coomassie blue (a). In some reactions individual capsid proteins were omitted. Lane 1 contains HSV-1 capsids isolated from infected BHK cells as a protein standard, and lane 2 contains a reaction mixture with all four recombinant proteins. Control HSV-1 capsids from infected cells contain processed scaffolding protein (VP22a), while in vitro assembly reactions contain uncleaved scaffolding protein (pre-VP22a). Lane 3 contains ALL minus VP23, lane 4 contains ALL minus VP19C, and lane 5 contains ALL minus VP23 and VP19C. Two identical gels were blotted and probed with polyclonal antiserum specific for VP19C (b) or VP23 (c). One additional band is visible just below VP19C (panel c, lane 2); this band likely represents a breakdown product resulting from protease activity in the insect cell extract.

FIG. 2

Sedimentation analysis of triplex proteins and protein complexes. Cell extracts containing VP19C, VP23, or VP19C and VP23 were placed on top of 5 to 20% sucrose gradients and centrifuged for 18 h at 115,000 × g. Each gradient was fractionated, and the fractions were analyzed by SDS-PAGE and Western blotting. Protein intensity was determined with Imagequant; the values were normalized, and the relative intensity values were plotted against distance from the meniscus. BSA was included in each gradient as a standard. ——, VP19C; — . — . , VP23; … …, BSA.

FIG. 3

Schematic diagram illustrating the deletions made in the VP19C protein (a) and Western blot detection of VP19C deletion mutants from insect cell extracts (b) or immunoprecipitated assembly complexes (c). VP19C deletion mutants were constructed by cloning truncated genes into baculovirus vectors as described in the text. Proteins that contained deletions at the N terminus or C terminus were expressed in Sf9 cells. Whole-cell lysates were analyzed by SDS-PAGE followed by Western blotting with polyclonal antiserum specific for VP19C (b). Mutants were tested in assembly reactions that included VP5, pre-VP22a, VP23, and either full-length VP19C or one of the deletion mutants. Assembly products were precipitated with monoclonal antibody 6F10 and analyzed by Western blotting as described above (c).

FIG. 4

Electron micrographs of capsids formed by coinfection of Sf9 cells with recombinant baculoviruses expressing VP5, pre-VP22a, VP23, and either wild-type VP19C or one of the deletion mutants. Cell pellets were fixed and thin sectioned. Capsids (arrows) were observed only in coinfections containing wild-type VP19 (a), nd45 (b), or nd90 (c). Aberrant structures (double arrows) were also seen in infections containing nd90 (c). Bar, 250 nm.

FIG. 5

Sedimentation analysis of VP19C deletion mutants and complexes containing mutant proteins. Cell extracts containing mutant cd15 (a), nd105 (b), cd15 and VP23 (c), or nd105 and VP23 (d) were placed on top of 5 to 20% sucrose gradients and centrifuged for 18 h at 115,000 × g. Each gradient was fractionated and analyzed as described previously, with protein intensity plotted versus distance from the meniscus to show the location of protein peaks. BSA was included in each gradient as a standard. ——, cd15; — . — . , nd105; … …, BSA.

FIG. 6

Proposed sequence of events in the HSV-1 capsid assembly pathway, based on intermediates observed in the in vitro assembly system. The major capsid protein and the scaffold protein interact in the cytoplasm, forming VP5–pre-VP22a complexes that are localized to the nucleus (16). VP19C and VP23 interact to form triplexes, which then move to the nucleus. Assembly occurs by interaction of the two types of protein complexes, forming arc-like partial capsids that grow into a procapsid shell. Finally, the spherical procapsid matures by transformation into the icosahedral capsid. In vivo, this transformation would be expected to be accompanied by packaging of viral DNA.